Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFβ) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFβ. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.

中文翻译：

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人肝星状细胞释放的胶原蛋白需要维生素 C 并被羟化酶抑制有效阻断

肝纤维化的特点是细胞外基质蛋白的积累，主要由胶原蛋白组成。肝星状细胞 (HSC) 通过分泌胶原蛋白介导肝纤维化。维生素 C（抗坏血酸）是脯氨酰羟化酶的辅助因子，可修饰分泌途径上新合成的胶原蛋白。与大多数动物不同，人类不能合成抗坏血酸，其在肝纤维化中的作用仍不清楚。在这里，我们确定了抗坏血酸和脯氨酰羟化酶抑制对人 HSC 产生和分泌胶原蛋白的影响。用抗坏血酸、转化生长因子-β (TGFβ) 和/或泛羟化酶抑制剂二甲基草酰甘氨酸 (DMOG) 处理原代人 HSC (p-hHSC) 和人 HSCscell line LX-2。通过 RT-qPCR (COL1A1)、蛋白质印迹、和免疫荧光显微镜。通过蛋白质印迹法测定胶原蛋白-I 和 HPLC 测定羟脯氨酸浓度，测定培养基中的胶原蛋白分泌。在健康和肝硬化人体组织中对编码钠依赖性维生素 C 转运蛋白 1 和 2 (SVCT1/SVCT2) 的溶质载体家族 23 成员 1 和 2 (SLC23A1/SLC23A2) 的表达进行量化。在没有抗坏血酸的情况下，胶原蛋白-I 在 p-hHSCs 和 LX-2 细胞中积累，这被 TGFβ 增强。抗坏血酸联合处理强烈促进了 I 型胶原蛋白的排泄并提高了细胞外羟脯氨酸的浓度，而不会影响 I型胶原蛋白（COL1A1）的 mRNA 水平。即使在抗坏血酸存在的情况下，DMOG 也能抑制 I 型胶原蛋白的释放，并抑制 COL1A1 和 α-平滑肌肌动蛋白 (αSMA/ACTA2) mRNA 水平，在缺氧条件下也是如此。肝细胞表达两种抗坏血酸转运蛋白，而 p-hHSCs 和 LX-2 表达唯一的 SVCT2，其在肝硬化肝脏中选择性增强。人类 HSC 依靠抗坏血酸来有效分泌 I 型胶原蛋白，这可以被羟化酶拮抗剂有效阻断，揭示了治疗肝纤维化的新治疗靶点。